A fundamental area of inquiry in pharmacology and medicine is the determination of ligand-receptor interactions. The pharmacological basis of drug action, at the cellular level, is quite often the consequence of non-covalent interactions between therapeutically relevant small organic molecules and high affinity binding proteins within a specific cell type. These small organic ligands may function as agonists or antagonists of key regulatory events which orchestrate both normal and abnormal cellular functions. For years the pharmaceutical industry's approach to discovering such ligands has been one of the random screening of thousands of small molecules in specific in vitro and in vivo assays to determine a potent lead compound for their drug discovery efforts. This lead compound often exerts very well-defined effects with regard to cell function (e.g. inhibition of cytokine production or DNA replication) but its mechanism of action at the molecular (ligand-protein interaction) level remains elusive. There is an unmet need for a general and efficient method to identify the cellular targets for these pharmacological agents so as to accelerate the search for novel drugs both at the basic and applied levels of research.
At this time, no efficient methodologies exist for rapidly identifying a biological target such as a protein for a particular small molecule ligand. Existing approaches include the use of affinity chromatography, radio-labeled ligand binding and photoaffinity labeling in combination with protein purification methods to detect and isolate putative target proteins. This is followed by cloning of the gene encoding the target protein based on the peptide sequence of the isolated target. These approaches depend on the abundance of the putative target protein in the sample and are laborious and painstaking. There is no existing technology allowing for the direct identification of the cDNA encoding a target for a given ligand.
Similarly, no efficient general approach exists for identifying a small molecule capable of binding any selected cell target regardless of its biological function. Fowlkes et al. and Broach et al (WO 94/23025, WO 95/30012) developed a screening assay for identifying molecules capable of binding cell surface receptors so as to activate a selected signal transduction pathway. These references describe the modification of selected yeast signaling pathways so as to mimic steps in the mammalian signaling pathway. This latter approach is specific for certain signaling pathways and has limited utility for broadly discovering small molecules that interact with any cellular target.
Recently, a yeast genetic screening method has been developed for specifically identifying protein-protein interactions in an in vivo system. This assay is known as the Yeast Two-Hybrid system. (see FIG. 1, U.S. Pat. No. 5,468,614; and Yang et al. (1995) Nucleic Acid Research 23, 1152-1156). The yeast Two-Hybrid system relies on the interaction of two fusion proteins to bring about the transcriptional activation of a reporter gene such as E.coli derived .beta.-galactosidase (Lac Z). One fusion protein comprises a preselected protein fused to the DNA binding domain of a known transcription factor. The second fusion protein comprises a polypeptide from a cDNA library fused to a transcriptional activation domain. In order for the reporter gene to be activated, the polypeptide from the cDNA library must bind directly to the preselected target protein. Yeast cells harboring an activated reporter gene can be differentiated from other cells and the cDNA encoding for the interacting polypeptides can be easily isolated and sequenced. However, this assay is unsuited for screening small molecule-protein interactions because it relies solely on genetically encoded fusion proteins.
There is an unmet need for a general screening method to determine the interaction between small molecules and protein targets so as to identify new drugs that are capable of specific therapeutic effects in a variety of disease states as well as to identify agonists and antagonists that may interfere or compete with the binding of the small molecules for these targets.